Alphaviruses, including Venezuelan Equine Encephalitis (VEEV) and Chikungunya (CHIKV) viruses, pose a serious threat to human health around the world. VEEV has been developed as a biological weapon and is still considered a potential bioterrorism agent. A CHIKV epidemic in India in 2007 sickened millions and subsequently spread through Africa and into Europe. In December 2013 CHIKV transmission from mosquitoes to humans was confirmed in the Caribbean, demonstrating that CHIKV has emerged in the Western Hemisphere and that the United States is at risk. Despite the dangers that alphaviruses pose to human health there is currently no antiviral treatment for any alphavirus infection. The major reason no treatments are available is the lack of high-throughput screening (HTS)-amenable assays that target critical aspects of alphavirus replication. We previously developed HTS assays for flavivirus RNA capping inhibitors and used the assays to identify molecules that block flavivirus replication, demonstrating that chemical inhibition of virl RNA capping effectively blocks viral replication. To address the pressing need for alphavirus-specific therapeutics, we propose adapting our capping inhibitor approach to alphaviruses and employing this novel HTS screening assay to identify inhibitors of alphavirus RNA capping. Alphaviruses cap their genomes primarily via the action of their nsP1 RNA capping protein. The nsP1 capping enzyme binds GTP and transfers a GMP to RNAs to form the RNA cap. Therefore, molecules that specifically mimic and block GTP binding by nsP1 may serve as potent inhibitors of viral replication, as we have shown for the flaviviruses. We have purified VEEV nsP1 protein and shown that it is enzymatically active and able to bind GTP in an HTS-amenable fluorescence polarization assay. This project will capitalize on these results and establish robust HTS assays for both VEEV and CHIKV nsP1 proteins, allowing us to identify both species- and genus-specific anti-alphavirus inhibitors. Aim 1: Optimize expression and purification parameters for VEEV and CHIKV nsP1 proteins and establish optimal parameters for screening. Aim 2: Screen 16,200 compounds housed at Colorado State University and characterize screening hits with orthogonal and secondary biochemical and cell-based assays. A key component of this project is our ongoing collaboration with the CSU Colorado Center for Drug Discovery, which will provide critical medicinal chemistry support, help prioritize active chemical series, and assist in streamlining our assay development efforts. This project will place us in an excellent position to establish alphavirus RNA capping as a potent target for antiviral therapeutics and set the stage for subsequent full-scale screening and preclinical development efforts.